Protein C is an important component of the coagulation system that has strong anticoagulant activity. In its active form it is a serine protease that proteolytically inactivates Factors Va and VIIIa.
Human protein C (hPC) is a 62 kD, disulfide-linked heterodimer consisting of a 25 kD light chain and a 41 kD heavy chain which circulates as an inactive zymogen in plasma. At the endothelial cell surface it is activated to activated protein C (APC) by limited thrombin proteolysis in the presence of thrombomodulin; cleavage of an Arg-Leu bond in the amino terminal portion of the heavy chain releases a 12 amino acid peptide. See generally Gardiner & Griffin in PROGRESS IN HEMATOLOGY, Vol. XIII at page 265-278 (Brown, Grune and Stratton, Inc. 1983).
Several regions of the molecule have important implications for function as an anticoagulant in the regulation of hemostasis. The amino terminal portion of the light chain contains the nine y-carboxyglutamic acid (Gla) residues required for calcium-dependent membrane binding and functional activation. Another post-translational modification is .beta.-hydroxylation of aspartic acid reside 71, possibly required for calcium-dependent membrane binding which is independent of the binding activity of the Gla regions.
There are a variety of clinical situations for which protein C may prove beneficial. It may serve as replacement therapy in homozygous deficient infants suffering from purpura fulminans neonatalis. Other conditions include patients with a previous history of warfarin-induced skin necrosis who must have additional warfarin therapy, heparin-induced thrombocytopenia, septic shock for prevention of intravascular coagulation and organ damage, and for fibrinolytic therapy, as protein C can protect tPA from plasma inhibitor proteins. Table 1 represents one estimate of the number of individual cases of several clinical syndromes which might be treated by purified protein C. Because there has not been sufficient material available from plasma for clinical trials until recently, these data are necessarily based on an incomplete assessment of the therapeutic potential for protein C.
TABLE 1 PARTIAL ESTIMATE OF U.S. CLINICAL REQUIREMENTS FOR PROTEIN C AND ACTIVATED PROTEIN C Estimated Dose (mg) Per # Treatments Total U.S. Indication Treatment Per Year Req. (Kg) Septic Shock 5-50 120,000 0.6-6.0 Thrombolytic 10-100 800,000 8-80 Therapy** Hip Replacement 10-100 200,000 2-20 Homozygous 3 100 .times. 365* 0.10 Deficient Heterozygous 50 1,000 0.05 Deficient Total 10.8-106.2 *100 individuals in U.S. .times. 365 treatment/year **Referes to the use of APC, following thrombolytic therapy, to prevent the reformation of blood clots.
The gene for human protein C has been cloned and sequenced, as has bovine protein C gene. See Forster et al., Proc. Nat'l Acad. Sci. USA 82: 4673 (1985); U.S. Pat. No. 4,775,624. It is synthesized as an inactive precursor that undergoes several proteolytic events during the processes of secretion and activation. First, a signal sequence as proteolytically removed upon secretion. A second proteolytic event removes the dipeptide lys156 arg157, producing the inactive zymogen, a two chain disulfide bridged protein, consisting of a light chain of 155 amino acids and a heavy chain of 262 amino acids. The zymogen is activated by a final proteolytic event that removes residues 158-169, yielding active protein C, a serine protease with potent anticoagulant activity. Beckmann et al., Nucleic Acids Res. 13: 5233 (1985).
In addition to proteolytic processing, human protein C undergoes several post-translation modifications. Perhaps most salient among these modifications is the .gamma.-carboxylation of the first nine glutamic acid residues in protein C, by a vitamin K dependent enzyme. DiScipio & Davie, Biochemistry 18: 899 (1979). Gamma-carboxylation is required for anticoagulant activity, and is associated with Ca.sup.2+ -dependent membrane binding. The anticoagulant activity of protein C varies directly with the extent of .gamma.-carboxylation, and the highest levels of activity are achieved only when .gamma.-carboxylation of the sixth and seventh glutamic acid residues is effected. Zhang & Castellino, Biochemistry 29: 10829 (1990).
Protein C is also post-translationally modified by .beta.-hydroxylation of aspartic acid 71. Drakenberg et al., Proc. Nat'l Acad. Sci. USA 80: 1802 (1983). Beta-hydroxylation may be important to protein C activity. Although its function is not known it has been suggested that it may be involved in .gamma.-carboxyglutamic acid independent Ca.sup.2+ binding, and it may be required for full anti-coagulant activity.
Human protein C is also glycosylated. Kisiel, J. Clin. Invest. 64: 761 (1979). It contains four potential N-linked glycosylation sites, located at Asn97, Asn248, Asn313 and Asn329. The first three signals match the consensus Asn-X-Ser/Thr glycosylation sequences, and are actively glycosylated. There is an atypical glycosylation signal at Asn329, Asn-X-Cys-Ser. The Asn329 signal is glycosylated in bovine protein C, but it is not yet known if Asn329 is glycosylated in human protein C. Miletich et al., J. Biol. Chem. 265: 11397 (1990). The pattern and extent of glycosylation can alter the physiological activity of protein C.
Until recently, human protein C for experimental and therapeutic use was obtained exclusively from human plasma. Unfortunately, the quantity of protein that can be obtained from human serum is limited. Furthermore, products derived from human serum pose difficulties of reliability, purity and safety.
The expression of therapeutic proteins by recombinant DNA technology is an attractive alternative to plasma production of protein C, in that it eliminates the risk of potential contamination with blood-borne viruses and theoretically provides an unlimited supply of product. But the complexity of the post-translational modifications, as discussed above, has rendered problematic the production of commercially useable amounts of suitably active protein C by expression in a heterologous host.
In fact, it has not been possible to produce vitamin K-dependent proteins like protein C at sufficiently high levels in an active form, despite efforts to do so using a variety of expression systems. See Grinnell et al. in Volume 11 of ADVANCES IN APPLIED BIOTECHNOLOGY SERIES, Chapter 3 (Gulf Publishing Co.). In particular, any prospect for expressing protein C in mammary glands of a transgenic animal and secreting the protein into milk, see, e.g., U.S. Pat. No. 4,873,316 (1989), is clouded by the fact that protein C is normally synthesized in the liver. Even HepG2 cell lines derived from human liver produce aberrant forms of protein C. Marlar & Fair (1985).
In this regard, it has been observed that a mouse mammary epithelial cell line (C-127) transfected with a bovine papilloma virus (BPV) vector bearing the cDNA for human protein C expressed protein C that was only 30-40% active. Further analysis revealed that the protein C contained diminished levels of .gamma.-carboxyglutamic acid and little, if any, .beta.-hydroxyaspartic acid. Suttie et al., Thrombosis Res. 44: 129 (1986). These experiments indicate that mouse mammary epithelial cells cannot perform all of the post-translational modifications necessary for obtaining suitably active protein C, which in turn casts doubt on the likelihood of obtaining such protein C from the milk of a transgenic mammal.